There are currently intensive global research efforts aimed at increasing and modifying the accumulation of lipids, alcohols, hydrocarbons, polysaccharides, and other energy storage compounds in photosynthetic organisms, yeast, and bacteria through genetic engineering. Many improvements have been realized, including increased lipid and carbohydrate production, improved H2 yields, and the diversion of central metabolic intermediates into fungible biofuels. Photosynthetic microorganisms are attracting considerable interest within these efforts due to their relatively high photosynthetic conversion efficiencies, diverse metabolic capabilities, superior growth rates, and ability to store or secrete energy-rich hydrocarbons. Relative to cyanobacteria, eukaryotic microalgae possess several unique metabolic attributes of relevance to biofuel production, including the accumulation of significant quantities of triacylglycerol; the synthesis of storage starch (amylopectin and amylose), which is similar to that found in higher plants; and the ability to efficiently couple photosynthetic electron transport to H2 production. Although the application of genetic engineering to improve energy production phenotypes in eukaryotic microalgae is in its infancy, significant advances in the development of genetic manipulation tools have recently been achieved with microalgal model systems and are being used to manipulate central carbon metabolism in these organisms. It is likely that many of these advances can be extended to industrially relevant organisms. This review is focused on potential avenues of genetic engineering that may be undertaken in order to improve microalgae as a biofuel platform for the production of biohydrogen, starch-derived alcohols, diesel fuel surrogates, and/or alkanes.

Genetic Engineering of Algae for Enhanced Biofuel Production

https://www.cell.com/cell/pdf/S0092-8674(09)00148-2.pdf

RNA and Disease

Biofilm refers to the complex, sessile communities of microbes found either attached to a surface or buried firmly in an extracellular matrix as aggregates. The biofilm matrix surrounding bacteria makes them tolerant to harsh conditions and resistant to antibacterial treatments. Moreover, the biofilms are responsible for causing a broad range of chronic diseases and due to the emergence of antibiotic resistance in bacteria it has really become difficult to treat them with efficacy. Furthermore, the antibiotics available till date are ineffective for treating these biofilm related infections due to their higher values of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), which may result in in-vivo toxicity. Hence, it is critically important to design or screen anti-biofilm molecules that can effectively minimize and eradicate biofilm related infections. In the present article, we have highlighted the mechanism of biofilm formation with reference to different models and various methods used for biofilm detection. A major focus has been put on various anti-biofilm molecules discovered or tested till date which may include herbal active compounds, chelating agents, peptide antibiotics, lantibiotics and synthetic chemical compounds along with their structures, mechanism of action and their respective MICs, MBCs, minimum biofilm inhibitory concentrations (MBICs) as well as the half maximal inhibitory concentration (IC50) values available in the literature so far. Different mode of action of anti biofilm molecules addressed here are inhibition via interference in the quorum sensing pathways, adhesion mechanism, disruption of extracellular DNA, protein, lipopolysaccharides, exopolysaccharides and secondary messengers involved in various signaling pathways. From this study, we conclude that the molecules considered here might be used to treat biofilm-associated infections after significant structural modifications, thereby investigating its effective delivery in the host. It should also be ensured that minimum effective concentration of these molecules must be capable of eradicating biofilm infections with maximum potency without posing any adverse side effects on the host.

https://www.tandfonline.com/doi/pdf/10.1080/21505594.2017.1313372

Strategies for combating bacterial biofilms: A focus on anti-biofilm agents and their mechanisms of action

For more than 140 years, pollen tube guidance in flowering plants has been thought to be mediated by chemoattractants derived from target ovules. However, there has been no convincing evidence of any particular molecule being the true attractant that actually controls the navigation of pollen tubes towards ovules. Emerging data indicate that two synergid cells on the side of the egg cell emit a diffusible, species-specific signal to attract the pollen tube at the last step of pollen tube guidance. Here we report that secreted, cysteine-rich polypeptides (CRPs) in a subgroup of defensin-like proteins are attractants derived from the synergid cells. We isolated synergid cells of Torenia fournieri, a unique plant with a protruding embryo sac, to identify transcripts encoding secreted proteins as candidate molecules for the chemoattractant(s). We found two CRPs, abundantly and predominantly expressed in the synergid cell, which are secreted to the surface of the egg apparatus. Moreover, they showed activity in vitro to attract competent pollen tubes of their own species and were named as LUREs. Injection of morpholino antisense oligomers against the LUREs impaired pollen tube attraction, supporting the finding that LUREs are the attractants derived from the synergid cells of T. fournieri.

Defensin-like polypeptide LUREs are pollen tube attractants secreted from synergid cells

Engineering algae for biohydrogen and biofuel production

DNA gyrase is a type II topoisomerase that can introduce negative supercoils into DNA at the expense of ATP hydrolysis. It is essential in all bacteria but absent from higher eukaryotes, making it an attractive target for antibacterials. The fluoroquinolones are examples of very successful gyrase-targeted drugs, but the rise in bacterial resistance to these agents means that we not only need to seek new compounds, but also new modes of inhibition of this enzyme. We review known gyrase-specific drugs and toxins and assess the prospects for developing new antibacterials targeted to this enzyme.

/pdf/exploiting-bacterial-dna-gyrase-as-a-drug-target-current-586j1jrz1v.pdf

Exploiting bacterial DNA gyrase as a drug target: current state and perspectives.

MicroRNAs (miRNAs) are small RNAs, 21 to 22 nucleotides long, with important regulatory roles. They are processed from longer RNA molecules with imperfectly matched foldback regions and they function in modulating the stability and translation of mRNA. Recently, we and others have demonstrated that the unicellular alga Chlamydomonas reinhardtii, like diverse multicellular organisms, contains miRNAs. These RNAs resemble the miRNAs of land plants in that they direct site-specific cleavage of target mRNA with miRNA-complementary motifs and, presumably, act as regulatory molecules in growth and development. Utilizing these findings we have developed a novel artificial miRNA system based on ligation of DNA oligonucleotides that can be used for specific high-throughput gene silencing in green algae.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-313X.2008.03767.x

Highly specific gene silencing by artificial microRNAs in the unicellular alga Chlamydomonas reinhardtii

Nucleic acid analogs and mimics are commonly the modifications of native nucleic acids at the nucleobase, the sugar ring, or the phosphodiester backbone Many forms of promising nucleic acid analogs and mimics are available, such as locked nucleic acids (LNAs), peptide nucleic acids (PNAs), and morpholinos LNAs, PNAs, and morpholinos can form both duplexes and triplexes and have improved biostability They have become a general and versatile tool for DNA and RNA recognition LNA is a general and versatile tool for specific, high-affinity recognition of single-stranded DNA (ssDNA) and single-stranded RNA (ssRNA) LNA can be used for designing LNA oligoes for hybridization studies or as real time polymerase chain reaction probes in the form of Taqman probes LNA also has therapeutic and diagnostic applications PNA is another type of DNA analog with neutral charge The extreme stability of PNA makes it an ideal candidate for the antisense and antigene application PNA is used as probe for gene cloning, mutation detection, and in homologous recombination studies It was also used to design transcription factor decoy molecules for target gene induction Morpholino, another structural type, was devised to circumvent cost problems associated with DNA analogs It has become the premier knockdown tool in developmental biology due to its cytosolic delivery in the embryos by microinjection Thus, the nucleic acid analogs provide an advantage to design and implementation, therapies, and research assays, which were not implemented due to limitations associated with standard nucleic acids chemistry

Promising nucleic acid analogs and mimics: characteristic features and applications of PNA, LNA, and morpholino.

https://repository.up.ac.za/bitstream/handle/2263/21873/Karkare_Naphthoquinone(2013).pdf;sequence=1

The naphthoquinone diospyrin is an inhibitor of DNA gyrase with a novel mechanism of action.

Systemic life-threatening fungal infections represent a significant unmet medical need. Cell-based, phenotypic screening can be an effective means of discovering potential novel antifungal compounds, but it does not address target identification, normally required for compound optimization by medicinal chemistry. Here, we demonstrate a combination of screening, genetic, and biochemical approaches to identify and characterize novel antifungal compounds. We isolated a set of novel non-azole antifungal compounds for which no target or mechanism of action is known, using a screen for inhibition of Saccharomyces cerevisiae proliferation. Haploinsufficiency profiling of these compounds in S. cerevisiae suggests that they target Erg11p, a cytochrome P450 family member, which is the target of azoles. Consistent with this, metabolic profiling in S. cerevisiae revealed a buildup of the metabolic intermediates prior to Erg11p activity, following compound treatment. Further, human cytochrome P450 is also inhibited in in vitro assays by these compounds. We modeled the Erg11p protein based on the human CYP51 crystal structure, and in silico docking of these compounds suggests that they interact with the heme center in a manner similar to that of azoles. Consistent with these docking observations, Candida strains carrying azole-resistant alleles of ERG11 are also resistant to the compounds in this study. Thus, we have identified non-azole Erg11p inhibitors, using a systematic approach for ligand and target characterization.

Shantanu Karkare

Papers

Exploiting bacterial DNA gyrase as a drug target: current state and perspectives.

Highly specific gene silencing by artificial microRNAs in the unicellular alga Chlamydomonas reinhardtii

Promising nucleic acid analogs and mimics: characteristic features and applications of PNA, LNA, and morpholino.

The naphthoquinone diospyrin is an inhibitor of DNA gyrase with a novel mechanism of action.

An Integrated Approach for Identification and Target Validation of Antifungal Compounds Active against Erg11p